Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

Abstract

Abbreviations used: dSTORM, direct stochastic optical reconstruction microscopy; MAL, myelin and lymphocyte; Syt7, synaptotagmin-7; TIRF, total internal reflection fluorescence.M.-I. Thoulouze and A. Alcover contributed equally to this paper. Fig. 1, D and H). LAT vesicular compartment co-localized with Rab27a (35%) and Rab37 (30%; Fig. 1, M and N), two Rab molecules known to regulate cytokine and cytotoxic granule exocytosis in CD4 + and CD8 + T cells, respectively (Stinchcombe et al., 2001;Huse et al., 2006). TCR peripheral vesicles colocalized to the fast recycling Rab4b compartment (8%), whereas its core co-localized to Rab3d-(25%) and Rab8b-regulated (30%) exocytic compartments (Huse et al., 2006;Fukuda, 2008; Fig. 1, Q-S). Noteworthy, the secretory v-SNARE protein Ti-VAMP (Chaineau et al., 2009) co-localized with both LAT (30%) and TCR (30%) vesicular compartments (Fig. 1, O and W). Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse LAT and TCR vesicular release is regulated by calcium and synaptotagmin-7The traffic regulators present in LAT and TCR vesicles (Fig. 1) have been reported to be involved in stimulus-induced exocytosis at cytotoxic synapses (Ménager et al., 2007; MarcetPalacios et al., 2008). To pinpoint the molecular mechanism regulating Lck, LAT, and TCR release from vesicular compartments, we assessed the role of calcium, a known regulator of stimulus-dependent exocytosis (Stojilkovic, 2005;Pang and Südhof, 2010). To increase cytosolic calcium levels, we treated CD4 T cells either with thapsigargin or ionomycin. To accurately quantify Lck, LAT, and TCR distribution between intracellular vesicles and the plasma membrane, we stained for surface CD2 and then used it to automatically segment the plasma membrane. The resulting automated mask delimiting the internal and external outlines of the plasma membrane allowed us to discriminate and quantify the subcellular distribution (plasma membrane versus vesicular compartment) of our proteins of interest (materials and methods; unpublished data). We found LAT and TCR distribution to the vesicular compartment to be higher than anticipated by others (Bonello et al., 2004), 75% (Fig. 2, A, B, H, and I). In fact, through plasma membrane segmentation, we were able to distinguish plasma membrane-resident LAT and TCR from their intracellular compartments and subcortical vesicles, within the limits of the confocal microscopy resolution (unpublished data). Lck displayed a higher plasma membrane distribution with 25% in intracellular vesicles (Fig. 2, C and J; and not depicted).Consistent with LAT localization in Rab27a and Rab37 exocytic compartments, increased cytosolic calcium led to a clear release of its vesicular compartment into the plasma membrane (Fig. 2, A, I, and O; and not depicted). In agreement with TCR-segregated distribution into a Rab3d and Rab8b exocytic compartment and also into Rab4b rapid recycling endosomes, cytosolic calcium increase induced an inc...

Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium

“…Interestingly, WASH deficient T cells show an aberrant traffic not only of the TCR, but also of other receptors, including CD28, lymphocyte function-associated antigen 1 (LFA-1), TfR and GLUT1 (34), which identifies this adaptor as a non-exclusive regulator of receptor recycling in T cells. Other Rab GTPases that may participate in TCR recycling have been recently identified by Soares et al (35) who, based on a screening of a panel of Rabs that had been previously implicated in exocytic endosomal traffic, provided evidence that endosomal CD3ζ colocalizes with Rab3d and Rab8b (Figure 1).…”

“…Moreover, the tetanus toxin insensitive v-SNARE VAMP-7, which is coupled to the calcium sensor synaptotagmin-7, has been shown localize to subsynaptic vesicles at TCR activation sites, promoting the recruitment and docking of endosomal LAT to target membranes (36). Interestingly, the high resolution microscopy analysis carried out by Soares et al (35) on the endosomal pools of LAT and Lck showed that, while associated with recycling endosomes, similar to CD3ζ, these molecules are localized in distinct endosome subpopulations marked by a specific complement of Rabs, with LAT colocalizing with Rab27a and Rab37, and Lck with Rab11b as well with myelin and lymphocyte protein (MAL), a lipid raft associated protein known to be implicated in Lck transport to the plasma membrane. Moreover vesicular CD3ζ and LAT, but not Lck, were found to be associated with VAMP-7.…”

“…Moreover vesicular CD3ζ and LAT, but not Lck, were found to be associated with VAMP-7. These specific associations translate into functionally distinct traffic pathways, with MAL specifically required for the release of Lck, and calcium and synaptotagmin-7 for CD3ζ and LAT (35). While the function of the Rabs identified in the different pathways needs as yet to be addressed, these results demonstrate that endosomes that carry recycling receptors or signaling mediators to the IS are a mosaic of endosomes which traffic to the IS with the assistance of a specific complement of Rabs and SNAREs, generating 'nanoterritories' (35) where signaling is coordinated to promote T cell activation.…”

Meeting after meeting: 20 years of discoveries by the members of the Exocytosis–Endocytosis Club

“…Vesicles transporting the signalling adapter LAT have been reported to either dock and stay as subsynaptic vesicles or fuse with the plasma membrane, driving the clustering of LAT at the synapse (reviewed in Niedergang et al, 2016). The protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments as demonstrated by Andres Alcover (Soares et al, 2013). This definitive work established that tetanus-toxin sensitive SNARES are crucial for the polarised recruitment of TCRs at the immunological synapse (Das et al, 2004).…”

The Deleterious Effects of Oxidative and Nitrosative Stress on Palmitoylation, Membrane Lipid Rafts and Lipid-Based Cellular Signalling: New Drug Targets in Neuroimmune Disorders

“…This phosphorylation of ITAM acts to recruit the ZAP-70 tyrosine kinase normally located in the cytosol to the plasma membrane, whose main substrate is the transmembrane protein LAT otherwise described as linker for activation of T cells [28,126]. The subsequent phosphorylation of LAT leads to the assembly of multiple proteins, such as Grb-2 and Gads [28,127]. These LAT enucleated protein complexes mediate downstream pathways that cause alterations in gene transcription and cell morphology [28,128].…”